Screening And Identification Of Entry Inhibitors Against Lassa Virus From An FDA-approved Drug Library

Lassa virus?LASV?belongs to genus Mammarenavirus,family Arenaviridae,and is prioritized as a category A bioterrorism agent by the US Centers for Disease Control and Prevention as it causes fatal Lassa fever in humans.The natural host of the LASV is the rodent Mastomys natalensis.Human infections occur through contact with infected rodents or inhalation of aerosolized virus from contaminated rodent excrements or secretions,and this disease is also directly transmitted from human to human.LASV is mainly endemic in West Africa and infects 300,000 to 500,000 people annually.The mortality rate among hospitalized patients is up to 70%and the survivors often have deafness and other severe sequelae.Up to now,there are no FDA-approved LASV drugs or vaccines and the current treatment can only use the broad-spectrum antiviral drug ribavirin,which is effective only when used in early infection.Therefore,there is an urgent need to develop an effective and specific drug against LASV.Virus entry is an attractive target for antiviral drugs because it is the first crucial step in the viral life cycle.Here we generated pseudotype?LASVpv?and recombinant?LASVrv?vesicular stomatitis viruses,which enveloped the native LASV glycoprotein complex on the surface of the virus particles and thereby mimicked the LASV entry process in a Biosafety Level 2 laboratory.These viruses carried the reporter genes and can be used in high throughput screen of compound libraries.We performed a high throughput screen from an FDA-approved drug library and identified two drugs that specifically inhibited the entry of LASV:lacidipine and phenothrin,with the IC₅₀ values of 2.6?M and 5.3?M,respectively.Mechanism of action study revealed that both drugs inhibited LASV entry by blocking low-pH induced membrane fusion.In addition,lacidipine also had a direct virucidal activity against LASV.Adaptive mutant analysis uncovered that substitution of T40,located in ectodomain loop of SSP?stable signal peptide?,with K conferred LASV remarkable resistance to lacidipine.Further investigation showed T40K,T40R and T40D mutant viruses conferred resistance to lacidipine,while T40A mutant virus showed no resistance.Infection of T40K mutant virus exhibited similar growth curves as WT,while T40R and T40D produced less amounts of viruses.We also investigated the broad-spectrum antiviral activities of two candidate drugs against arenavirus,and it was demonstrated that lacidipine can strongly suppress Mopeia virus?MOPV?and Guanarito virus?GTOV?with IC₅₀ of 4.8 and 6.2?M,respectively,and phenothrin had an inhibitory effect on MOPV,GTOV and Chapare virus?CHPV?with IC₅₀ of 8.3,6.1and 8.0?M,respectively.Since the T40 is not conserved in GPC of GTOV,we speculated that some amino acid other than K40 contributed the sensitivity of GTOV to lacidipine.To further determined the viral target,we selected the adaptive mutant strain by serially passaging GTOVrv in the presence of lacidipine and found that the resistance mutations were V36M and V436A,which are located in the ectodomain of SSP and the transmembrane domain of GP2,respectively.The sensitivity of GTOV to Lacipipine was significantly reduced when both sites are changed.In this study,we identified two FDA-approved small molecule drugs,lacidipine and phenothrin,inhibited LASV entry at a micromolar range.Both drugs inhibited LASV entry by blocking GPC-mediated membrane fusion and exhibited broad-spectrum antiviral activity against arenavirus to some extent.Mechanism analysis suggested two drugs probably act to stabilize the prefusion GPC complex and block low-pH triggering of membrane fusion by interaction with SSP and GP2.Above all,the hit drugs identified in this study provide novel insights into the molecular mechanisms underlying LASV entry and potential new therapies for treatment of arenavirus infection and related disease.